JP4474336B2 - Oral titanium implant material and manufacturing method thereof - Google Patents
Oral titanium implant material and manufacturing method thereof Download PDFInfo
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- JP4474336B2 JP4474336B2 JP2005188707A JP2005188707A JP4474336B2 JP 4474336 B2 JP4474336 B2 JP 4474336B2 JP 2005188707 A JP2005188707 A JP 2005188707A JP 2005188707 A JP2005188707 A JP 2005188707A JP 4474336 B2 JP4474336 B2 JP 4474336B2
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Images
Description
本発明は、口腔チタンインプラント材とその製造方法に関し、より詳細には、チタンの表面をリン酸化プルランにより被覆して生体適合性を高めた、口腔チタンインプラント材とその製造方法に関する。 The present invention relates to an oral titanium implant material and a method for producing the same, and more particularly to an oral titanium implant material and a method for producing the same, in which the surface of titanium is coated with phosphorylated pullulan to improve biocompatibility.
チタンは、優れた耐食性、展延性、硬度を有する金属であり、また、他の金属と比べ、生体適合性がよいことから、口腔インプラント材として広く用いられている。しかしながら、従来の口腔チタンインプラント材は、これを適用した対象者によっては、チタンと骨との親和性不良のため、骨に埋入したチタンの撤去を余儀なくされるケースが見受けられる。この不具合を解消する手段として、チタン表面を酸化して、チタン表面に酸化チタン層やリン酸カルシウム系ガラス層を設けたり（特許文献１）、チタン表面にチタン粉末を焼結した連続孔を有する多孔構造物を付与したり、或いは、チタン表面をヒドロキシアパタイト加工したり、エチレン／ビニルアルコール共重合体を被覆したり（特許文献２）して、チタンの生体適合性を向上させる工夫がなされている。
しかしながら、これらの従来法によっても、チタンの生体適合性は不充分であり、より生体適合性の高い、チタン加工品およびこれを用いて得られる口腔チタンインプラント材の確立が鶴首されている。 However, even by these conventional methods, the biocompatibility of titanium is inadequate, and the establishment of a titanium processed product and an oral titanium implant material obtained using the titanium product having higher biocompatibility has been sought.
本発明の課題は、生体適合性の優れた口腔チタンインプラント材とその製造方法を提供することにある。 The subject of this invention is providing the oral titanium implant material excellent in biocompatibility, and its manufacturing method.
本発明者等は、上記課題を解決することを目的として、チタン表面を被覆する材料として、従来より、生体適合性に優れた素材として知られている多糖類を広く検索した。さらに、本発明者等は、プルランに着目し、プルランを用いてチタンの生体適合性を高める手段について、鋭意研究した。 In order to solve the above-mentioned problems, the present inventors have extensively searched for polysaccharides that have been conventionally known as materials excellent in biocompatibility as materials for coating titanium surfaces. Furthermore, the present inventors paid attention to pullulan, and conducted earnest research on means for enhancing the biocompatibility of titanium using pullulan.
その結果、プルランはそのままでは、チタンに対する接着性が不十分であり、これをそのままチタン表面に被覆したのでは、チタン表面から容易に剥離してしまい、所期の目的が達成できないことが判明した。そこで、本発明者等は、プルランのチタンへの接着性を高めることを目的として、プルランにリン酸及び／又はポリリン酸を結合させてリン酸化プルランとしたところ、チタンへの接着性が著しく高まることを見出した。この知見に基づき、当該リン酸化プルランにより、チタン表面の一部または全部を被覆して得られるチタンを用いて得られる口腔チタンインプラント材は、本発明の所期の目的を達成し得ることを見出し、本発明を完成した。 As a result, it was found that the pullulan as it was had insufficient adhesion to titanium, and if this was directly coated on the titanium surface, it was easily peeled off from the titanium surface and the intended purpose could not be achieved. . Accordingly, the present inventors have made phosphoric pullulan by binding phosphoric acid and / or polyphosphoric acid to pullulan for the purpose of enhancing the adhesiveness of pullulan to titanium. I found out. Based on this finding, it was found that an oral titanium implant material obtained by using titanium obtained by coating a part or all of the titanium surface with the phosphorylated pullulan can achieve the intended purpose of the present invention. The present invention has been completed.
すなわち、本発明は、表面の一部または全部をリン酸化プルランにより被覆してなる生体適合性を高めた、口腔チタンインプラント材、及びチタンの表面の一部または全部をリン酸化プルランにより被覆する工程を含む、口腔チタンインプラント材の製造方法、及び、生体適合性に優れた口腔チタンインプラント材とその製造方法を提供することにより、上記課題を解決するものである。 That is, the present invention relates to an oral titanium implant material with improved biocompatibility formed by coating part or all of the surface with phosphorylated pullulan, and a step of coating part or all of the surface of titanium with phosphorylated pullulan. The above-mentioned problems are solved by providing a method for producing an oral titanium implant material, including an oral titanium implant material excellent in biocompatibility and a method for producing the same.
チタン表面の一部または全部を、プルランにリン酸及び／又はポリリン酸を結合させた、リン酸化プルランにより被覆することにより、生体適合性に優れたチタンを工業的に安価に製造することができる。本明細書においては、上記したリン酸化プルランを被覆したチタンを、単に、「表面処理チタン」と言う場合がある。斯かる表面処理チタンは、生体適合性に優れていることから、口腔チタンインプラント材としての優れた用途を有する。 By covering part or all of the titanium surface with phosphorylated pullulan in which pullulan and / or polyphosphoric acid are bound to pullulan, titanium having excellent biocompatibility can be produced industrially at low cost. . In the present specification, titanium coated with the above-described phosphorylated pullulan may be simply referred to as “surface-treated titanium”. Since such surface-treated titanium is excellent in biocompatibility, it has an excellent use as an oral titanium implant material.
本発明のリン酸化プルランの製造に用いるプルランは、グルコース３分子がα−１，４結合したマルトトリオースが、α−１，６結合で連なった多糖類であって、従来より、食品、化粧品、医薬品、農林水産品、化学薬品等の分野に広く用いられている。プルランは、生体に適用したとき、生体内の酵素の作用を受けて、徐々に分解・吸収されることから、生体適合性に優れた、安全な材料として知られている。プルランは、アミロース、アミロペクチン、グリコーゲンとは違って、生体内のアミラーゼで急速に分解されることがなく、生体内のα−グルコシダーゼなどによって緩慢に分解される特徴を有している。更に、プルランは、加熱又は適宜溶媒と混合することにより、容易にゲル状にすることができる特徴をも有している。ゲル状にしたプルランは、蛋白質や酵素などの生理活性物質を容易に取り込む物性を有し、かつ、取り込んだ生理活性物質を緩慢に放出することができる。また、プルランと生理活性物質とを適宜方法により化学的に結合させて複合体とした場合にも、取り込んだ生理活性物質を緩慢に放出させることができる利点を有する。 The pullulan used in the production of the phosphorylated pullulan of the present invention is a polysaccharide in which maltotriose in which three molecules of glucose are α-1,4 linked are linked by α-1,6 bonds. Widely used in fields such as pharmaceuticals, agriculture, forestry and fishery products, and chemicals. Pullulan is known as a safe material with excellent biocompatibility because it is gradually decomposed and absorbed by the action of enzymes in the living body when applied to a living body. Unlike amylose, amylopectin, and glycogen, pullulan is not rapidly degraded by amylase in vivo, but has a characteristic of being slowly degraded by α-glucosidase in vivo. Furthermore, pullulan has a characteristic that it can be easily gelled by heating or mixing with a solvent as appropriate. Gelled pullulan has physical properties that easily take in physiologically active substances such as proteins and enzymes, and can slowly release the incorporated physiologically active substance. In addition, even when pullulan and a physiologically active substance are chemically combined by an appropriate method to form a complex, there is an advantage that the incorporated physiologically active substance can be slowly released.
本発明で用いるプルランとしては、通常、数平均分子量が１０，０００乃至５，０００，０００ダルトンのプルランが好適に用いられる。プルランの数平均分子量は、分子量標準試料として市販されているプルラン（株式会社林原商事販売）を用いる、公知の高速液体クロマトグラフィー法により求めることができる。 As the pullulan used in the present invention, usually a pullulan having a number average molecular weight of 10,000 to 5,000,000 daltons is preferably used. The number average molecular weight of pullulan can be determined by a known high performance liquid chromatography method using pullulan (sold by Hayashibara Shoji Co., Ltd.) commercially available as a molecular weight standard sample.
本発明のリン酸化プルランの製造に用いるリン酸としては、オルトリン酸、オルトリン酸カリウム、オルトリン酸ナトリウム、及びポリリン酸ナトリウムから選ばれる１種又は２種以上を好適に用いることができる。 As phosphoric acid used for the production of the phosphorylated pullulan of the present invention, one or more selected from orthophosphoric acid, potassium orthophosphate, sodium orthophosphate, and sodium polyphosphate can be suitably used.
本発明のリン酸化プルランを製造するに際し、プルランとリン酸とを結合させる方法は、上記リン酸とプルランとを化学的に結合させることのできる方法であれば特に制限はない。一般的には、プルランを精製水に溶解し、得られるプルラン水溶液にリン酸を加え、混合した後、減圧乾燥して、水分を、通常、３０質量％以下、好適には、２０質量％以下、より好適には、１０質量％以下にして、通常、８０℃以上、好適には、１００℃乃至２００℃の高温下で、通常、５分間以上、好適には、１０分間乃至１０時間加熱することにより、リン酸とプルランとを化学的に結合させて、本発明で用いるリン酸化プルランを得ることができる。前記プルラン水溶液におけるプルラン濃度は、通常、５質量％以上、好適には、１０質量％以上、より好適には、１５〜４０質量％とする。プルラン水溶液に添加するリン酸の量は、プルラン質量に対し、通常、等量以上、好適には、１．５倍量以上、より好適には、２倍量以上とする。上記方法により得られるリン酸化プルランを精製する方法としては、例えば、溶媒沈殿法、透析法、電気透析法、イオン交樹脂やゲル濾過樹脂などを用いるカラムクロマトグラフィーなどを例示でき、これらの方法を適宜組み合わせて、残存する遊離のリン酸や未反応のプルランを除去して、リン酸化プルランを精製することができる。更に、必要に応じて、上記の方法により精製したリン酸化プルランを活性炭や高性能濾過によってパイロジェンを除去し、高純度リン酸化プルランとすることも随意である。 In producing the phosphorylated pullulan of the present invention, the method for bonding pullulan and phosphoric acid is not particularly limited as long as it is a method capable of chemically bonding the phosphoric acid and pullulan. In general, pullulan is dissolved in purified water, phosphoric acid is added to the resulting aqueous solution of pullulan, mixed and then dried under reduced pressure, and the water content is usually 30% by mass or less, preferably 20% by mass or less. More preferably, it is 10% by mass or less, and it is usually heated at a high temperature of 80 ° C. or higher, preferably 100 ° C. to 200 ° C., usually for 5 minutes or longer, preferably 10 minutes to 10 hours. Thus, phosphoric acid and pullulan can be chemically bonded to obtain the phosphorylated pullulan used in the present invention. The pullulan concentration in the aqueous pullulan solution is usually 5% by mass or more, preferably 10% by mass or more, and more preferably 15 to 40% by mass. The amount of phosphoric acid added to the aqueous pullulan solution is usually equal to or greater than the mass of pullulan, preferably 1.5 times or more, more preferably 2 times or more. Examples of the method for purifying the phosphorylated pullulan obtained by the above method include a solvent precipitation method, a dialysis method, an electrodialysis method, column chromatography using an ion-exchange resin or a gel filtration resin, and the like. The phosphorylated pullulan can be purified by appropriate combination to remove the remaining free phosphoric acid and unreacted pullulan. Furthermore, if necessary, the phosphorylated pullulan purified by the above-mentioned method is optionally activated by removing pyrogen by activated carbon or high performance filtration to obtain a high purity phosphorylated pullulan.
本発明のリン酸化プルラン中のリン酸含量は、本発明の所期の目的を達成するために、リンとして、０．０１質量％以上、好適には、０．０１質量％乃至５．０質量％が望ましく、その含量は、下記の方法で測定することができる。 In order to achieve the intended purpose of the present invention, the phosphoric acid content in the phosphorylated pullulan of the present invention is 0.01 mass% or more, preferably 0.01 mass% to 5.0 mass as phosphorus. % Is desirable, and its content can be measured by the following method.
即ち、リン酸化プルラン試料を１２０℃で５時間、真空乾燥し、得られた乾燥物１０ｇを秤量してビーカーに入れ、電気加熱器上で予備灰化した後、５００℃の電気炉中で灰化する。放冷後、灰に１８％塩酸水溶液３ｍｌを加え、水浴上で蒸発乾固する。さらに、１８％塩酸水溶液２ｍｌを加え、ビーカーの開口部を時計皿で覆い、２００℃のホットプレート上で３０分間加熱した後、ろ紙でろ過し、ろ液を回収する。さらに、精製水１０ｍｌを用いてビーカー内壁と、ろ紙を洗浄し、洗浄液を回収する。この洗浄操作をさらに２回繰り返し、洗浄水を回収する。最終的に、ろ液と洗浄水とを集め、精製水を加えて全量を２００ｍｌとする。次いで、本溶液中のリン酸含量をモリブデンブルー法を用いて定量する。 That is, a phosphorylated pullulan sample was vacuum-dried at 120 ° C. for 5 hours, and 10 g of the obtained dried product was weighed and placed in a beaker, pre-ashed on an electric heater, and then ashed in an electric furnace at 500 ° C. Turn into. After cooling, add 3 ml of 18% aqueous hydrochloric acid solution to the ash and evaporate to dryness on a water bath. Furthermore, 2 ml of 18% hydrochloric acid aqueous solution is added, the opening of the beaker is covered with a watch glass, heated on a hot plate at 200 ° C. for 30 minutes, filtered through filter paper, and the filtrate is recovered. Further, the inner wall of the beaker and the filter paper are washed with 10 ml of purified water, and the washing liquid is collected. This washing operation is further repeated twice to collect washing water. Finally, the filtrate and washing water are collected, and purified water is added to make a total volume of 200 ml. Subsequently, the phosphoric acid content in this solution is quantified using the molybdenum blue method.
本発明で用いるチタンは、歯科学的許容性のものであれば全て使用することができる。例えば、歯科用材料として製造され、販売されているチタン製品をそのまま、或いは、切除、整形又は成形等の加工処理を施した後、これらの表面の一部又は全体を研磨、洗浄したものであってもよい。 Any titanium can be used as long as it is dentally acceptable. For example, a titanium product manufactured and sold as a dental material may be used as it is or after it has been subjected to processing such as excision, shaping or molding, and a part or all of these surfaces are polished and washed. May be.
次に、本発明のリン酸化プルランをチタンに被覆する方法について述べる。その方法としては、予め、濃度０．１乃至１０モルの塩酸水溶液に１分間乃至１時間浸漬したチタンを、１質量％乃至３０質量％のリン酸化プルラン水溶液に浸漬、又は、その水溶液を塗布又は噴霧して、チタン表面にリン酸化プルランを付着させ、２０℃以上、好適には、２０乃至８０℃で乾燥することにより、リン酸化プルランをチタン表面に被覆することができる。チタン表面に被覆されるプルランの膜厚は、リン酸化プルラン水溶液の濃度、これをチタンに適用する量によって変化するが、通常、１μｍ以上、好適には、１〜８００μｍ、より好適には、５０〜５００μｍ、更に好適には、７０〜４００μｍとする。チタン表面に被覆したリン酸化プルラン膜は、室温下で長期間放置しても剥離することなく、チタン表面に安定に接着している。 Next, a method for coating the phosphorylated pullulan of the present invention on titanium will be described. As the method, titanium immersed in an aqueous hydrochloric acid solution having a concentration of 0.1 to 10 mol in advance for 1 minute to 1 hour is immersed in an aqueous solution containing 1% to 30% by weight of phosphorylated pullulan, or the aqueous solution is applied or applied. The phosphorous pullulan can be coated on the titanium surface by spraying to adhere the phosphorylated pullulan to the titanium surface and drying at 20 ° C. or higher, preferably 20 to 80 ° C. The film thickness of the pullulan coated on the titanium surface varies depending on the concentration of the phosphorylated pullulan aqueous solution and the amount of this applied to titanium, but is usually 1 μm or more, preferably 1 to 800 μm, more preferably 50 ˜500 μm, more preferably 70 to 400 μm. The phosphorylated pullulan film coated on the titanium surface is stably adhered to the titanium surface without being peeled even when left for a long time at room temperature.
また、リン酸化プルラン水溶液を調製するに際し、該水溶液中に適量の各種生理活性物質を加えて生理活性物質含有リン酸化プルラン水溶液とし、この水溶液を用いて上記の如くチタン表面を被覆し、生理活性物質含有リン酸化プルランをチタン表面に被覆することも随意である。前記生理活性物質含有リン酸化プルランを被覆したチタンを用いた場合、生体内において、生理活性物質を徐放させることができる。この際、生理活性物質として、骨芽細胞の形成と機能の制御に作用すると言われている活性型ビタミンＤ（１α，２５（ＯＨ）２Ｄ３）、副甲状腺ホルモン（ＰＴＨ）、エストロゲン（Ｅ２）、プロスタグランジンＥ２（ＰＧＥ２）などのホルモンを用い、斯かる生理活性物質を含有するリン酸化プルランをチタン表面に被覆し、これを生体に適用する場合には、チタンと骨芽細胞などとの初期接着性と、生体適合性とが顕著に向上する。 Further, when preparing a phosphorylated pullulan aqueous solution, an appropriate amount of various physiologically active substances is added to the aqueous solution to obtain a physiologically active substance-containing phosphorylated pullulan aqueous solution, and this aqueous solution is used to coat the titanium surface as described above. It is also optional to coat the titanium surface with the substance-containing phosphorylated pullulan. When titanium coated with the physiologically active substance-containing phosphorylated pullulan is used, the physiologically active substance can be gradually released in vivo. At this time, active vitamin D (1α, 25 (OH) 2 D 3 ), parathyroid hormone (PTH), estrogen (E), which are said to act on the control of osteoblast formation and function as physiologically active substances. 2 ), when hormones such as prostaglandin E 2 (PGE 2 ) are used, and phosphorylated pullulan containing such a physiologically active substance is coated on the surface of titanium and applied to a living body, titanium and osteoblast Initial adhesiveness with cells and biocompatibility are significantly improved.
本発明に係るリン酸化プルランに生理活性物質を保持させるに際し、リン酸化プルランをそのまま用いてもよいが、リン酸化プルランに、より強固に生理活性物質を保持させる目的で、塩化シアヌル、ブロムシアン、過ヨウ素酸などの活性化試薬を用いて、予め、リン酸化プルランを活性化させることも適宜である。生理活性物質を結合させたリン酸化プルランを被覆したチタンは、生体内において、当該プルランが緩慢に分解され、この分解に伴い、生理活性物質が緩慢に徐放される。 When the phosphorylated pullulan according to the present invention retains the physiologically active substance, the phosphorylated pullulan may be used as it is. It is also appropriate to activate phosphorylated pullulan in advance using an activating reagent such as iodic acid. Titanium coated with phosphorylated pullulan bound with a physiologically active substance is slowly decomposed in vivo, and the physiologically active substance is slowly and slowly released along with this decomposition.
以下、実験例と実施例とにより、本発明をより具体的に説明する。 Hereinafter, the present invention will be described in more detail with reference to experimental examples and examples.
＜リン酸化プルランの調製＞
プルラン（商品名『プルラン』、数平均分子量７０，０００ダルトン、株式会社林原商事販売）４５ｇに精製水２０５ｇを加え、攪拌しながら溶解した。このプルラン水溶液に、予め、オルトリン酸（和光純薬工業株式会社販売）を濃度１モル水溶液（ｐＨ５．５）となるように調製したリン酸水溶液１，０００ｇを加え、混合した後、全量を真空乾燥して水分９．７質量％とした。得られた乾燥物を１７０℃で５時間加熱してリン酸化プルランを得た後、これをミキサーで粉砕してリン酸化プルラン粉砕物を約１６０ｇ得た。この粉砕物を水３，８４０ｇに溶解した後、エタノール（純度９９％）３，０００ｇを加え、リン酸化プルランを沈殿させた。この沈殿物を遠心分離（５，０００ｒｐｍ、１５分間）して回収し、５０質量％エタノール水溶液２，０００ｇを用いて２回洗浄し、得られた沈殿物に精製水１５０ｇを加え、固形物を約６０ｇ含む、粗リン酸化プルラン水溶液２６３ｇを得た。この粗リン酸化プルラン含有水溶液にエタノール（純度９９％）２３０ｇを加え、リン酸化プルランを沈殿させた後、沈殿物を回収し、精製水３００ｇを加えて、再溶解し、再度、エタノール（純度９９％）２００ｇを加えてリン酸化プルランを沈殿させ、得られた沈殿物を６０℃で３時間減圧乾燥し、粉砕して、水分含量が５．６質量％である、精製リン酸化プルラン粉末を約４１ｇ得た。
<Preparation of phosphorylated pullulan>
205 g of purified water was added to 45 g of pullulan (trade name “Pullan”, number average molecular weight 70,000 daltons, sold by Hayashibara Shoji Co., Ltd.) and dissolved while stirring. To this pullulan aqueous solution, 1,000 g of phosphoric acid aqueous solution prepared in advance so that orthophosphoric acid (sales by Wako Pure Chemical Industries, Ltd.) is a 1 molar aqueous solution (pH 5.5) is added and mixed, and then the whole amount is vacuumed. It was dried to a moisture content of 9.7% by mass. The obtained dried product was heated at 170 ° C. for 5 hours to obtain phosphorylated pullulan, which was then pulverized with a mixer to obtain about 160 g of phosphorylated pullulan pulverized product. This ground product was dissolved in 3,840 g of water, and then 3,000 g of ethanol (purity 99%) was added to precipitate phosphorylated pullulan. The precipitate was collected by centrifugation (5,000 rpm, 15 minutes), washed twice with 2,000 g of 50% by mass ethanol aqueous solution, 150 g of purified water was added to the resulting precipitate, and the solid matter was recovered. 263 g of a crude phosphorylated pullulan aqueous solution containing about 60 g was obtained. After 230 g of ethanol (purity 99%) was added to the crude phosphorylated pullulan-containing aqueous solution to precipitate phosphorylated pullulan, the precipitate was recovered, 300 g of purified water was added and redissolved, and again ethanol (purity 99) %) 200 g was added to precipitate phosphorylated pullulan, and the resulting precipitate was dried under reduced pressure at 60 ° C. for 3 hours and pulverized to obtain purified phosphorylated pullulan powder having a water content of 5.6% by mass. 41 g was obtained.
上記方法により得られた精製リン酸化プルランを４０ｇ秤量し、精製水３６０ｇに溶解し、これに活性炭を２０ｇ加え、６０℃で１時間攪拌した後、濾過し、得られた濾液をデプスフィルター（商品名『ゼータプラス、Ｓ３０』、キュノ株式会社販売）で濾過した。得られた濾液（約３８０ｇ）にエタノール（純度９９％）を３８０ｇ加え、攪拌し、生成した沈殿物を遠心分離（７，０００ｒｐｍ、２０分間）して回収し、４０℃で１８時間真空乾燥し、粉砕して、水分含量が３．５質量％である、精製リン酸化プルラン標品（標品１）を３７ｇ得た。本標品１をエンドトキシン測定試薬（商品名『リムルスＪテストワコー』、和光純薬工業株式会社販売）で測定したところ、本標品１は、エンドトキシンを０．２５ＥＵ／ｍｌ未満含有し、パイロジェンは検出限界以下であり、安全性の極めて高い標品であった。本標品１の赤外吸収スペクトルを図１に、対照としてのプルラン（商品名『プルラン』、数平均分子量７０，０００ダルトン、株式会社林原商事販売）の赤外吸収スペクトルを図２に示す。
40 g of the purified phosphorylated pullulan obtained by the above method was weighed and dissolved in 360 g of purified water, 20 g of activated carbon was added thereto, stirred at 60 ° C. for 1 hour, filtered, and the filtrate obtained was filtered with a depth filter (product) The product was filtered with the name “Zeta Plus, S30” (available from Cuno Inc.). To the obtained filtrate (about 380 g), 380 g of ethanol (purity 99%) was added and stirred, and the resulting precipitate was collected by centrifugation (7,000 rpm, 20 minutes) and dried in vacuo at 40 ° C. for 18 hours. After pulverization, 37 g of a purified phosphorylated pullulan sample (standard sample 1) having a water content of 3.5% by mass was obtained. When this
＜リン酸含量の測定＞
実験例１の方法で得られた精製リン酸化プルラン標品（標品１）約１２ｇを１２０℃で５時間、真空乾燥し、得られた乾燥物１０ｇを秤量してビーカーに入れ、電気加熱器上で予備灰化した後、５００℃の電気炉中で灰化した。放冷後、灰化物に１８％塩酸水溶液３ｍｌを加え、水浴上で蒸発乾固した。さらに、１８％塩酸水溶液２ｍｌを加え、時計皿で覆い、２００℃のホットプレート上で３０分間加温した後、ろ紙でろ過し、ろ液を回収した。さらに、精製水１０ｍｌでビーカー内壁と、ろ紙とを洗浄し、その洗浄液を回収した。この洗浄操作をさらに２回繰り返し、洗浄水を回収した。ろ液と洗浄水とを集め、精製水を加えて全量を２００ｍｌとし、試験溶液とした。試験溶液１ｍｌを１００ｍｌ容メスフラスコに分取し、１％フェノールフタレインエタノール溶液を３滴加え、０．５％アンモニア水を微紅色を呈するまで加えた後、０．６％硝酸水溶液で中和した。水で全量を約７０ｍｌとした後、予め、調製しておいた０．１２質量％メタバナジン酸アンモニウム１．５質量％硝酸２．７質量％モリブデン酸アンモニウム混液２０ｍｌを加え、全量を１００ｍｌとした。３０分間放置した後、４１０ｎｍにおける本溶液の吸光度を測定した。なお、別途、リン酸一カリウム標準液を用いて、前記同様にその吸光度を測定し、その測定結果に基づいて作成した検量線に基づいて、リン含量を計算した。その結果、精製リン酸化プルラン標品（標品１）は、１ｇ当り、リンを約２４ｍｇ含んでいた。
<Measurement of phosphoric acid content>
About 12 g of the purified phosphorylated pullulan sample (sample 1) obtained by the method of Experimental Example 1 was vacuum-dried at 120 ° C. for 5 hours, and 10 g of the obtained dried product was weighed and placed in a beaker. After preliminary ashing above, ashing was performed in an electric furnace at 500 ° C. After allowing to cool, 3 ml of 18% hydrochloric acid aqueous solution was added to the incinerated product and evaporated to dryness on a water bath. Further, 2 ml of 18% hydrochloric acid aqueous solution was added, covered with a watch glass, heated on a hot plate at 200 ° C. for 30 minutes, filtered through filter paper, and the filtrate was collected. Furthermore, the inner wall of the beaker and the filter paper were washed with 10 ml of purified water, and the washing liquid was recovered. This washing operation was further repeated twice to collect washing water. The filtrate and washing water were collected and purified water was added to make a total volume of 200 ml, which was used as a test solution. Dispense 1 ml of the test solution into a 100 ml volumetric flask, add 3 drops of 1% phenolphthalein ethanol solution, add 0.5% aqueous ammonia until it shows a slight red color, and then neutralize with 0.6% nitric acid aqueous solution. did. The total amount was adjusted to about 70 ml with water, and 20 ml of a 0.12 mass% ammonium metavanadate 1.5 mass% nitric acid 2.7 mass% ammonium molybdate mixture prepared in advance was added to make the total volume 100 ml. After standing for 30 minutes, the absorbance of this solution at 410 nm was measured. Separately, the absorbance was measured in the same manner as described above using a monopotassium phosphate standard solution, and the phosphorus content was calculated based on a calibration curve created based on the measurement results. As a result, the purified phosphorylated pullulan sample (sample 1) contained about 24 mg of phosphorus per gram.
＜リン酸化プルランの調製＞
プルラン（商品名『プルランＰＩ２０』、株式会社林原商事販売、数平均分子量１１０，０００ダルトン）４５ｇに精製水を２０５ｇ加え、充分攪拌しながら溶解させた。このプルラン水溶液に、予め、ポリリン酸（和光純薬工業株式会社販売）を濃度１モル（ｐＨ５．５）となるように調製したポリリン酸水溶液を１，０００ｇ加え、混合した後、全量を真空乾燥して、水分含量が１０．２質量％となるまで乾燥した。得られた乾燥物を１７０℃で５時間加熱してリン酸化プルランを得た。得られたリン酸化プルランをミキサーで粉砕して、リン酸化プルラン粉砕物を約１３０ｇ得た。この粉砕物を精製水３，０００ｇに溶解した後、エタノール（純度９９％）を２，５００ｇ加え、リン酸化プルランを沈殿させた。得られた沈殿物を遠心分離（５，０００ｒｐｍ、１５分間）して回収し、更に、５０質量％エタノール水溶液２，０００ｇを用いて２回洗浄し、得られた沈殿物に精製水を１００ｇ加え、固形物を約２５ｇ含有する粗リン酸化プルラン含有水溶液を約１３０ｇ得た。この粗リン酸化プルラン含有水溶液にエタノール（純度９９％）を１３０ｇ加え、リン酸化プルランを沈殿させた後、沈殿物を回収し、精製水を１２０ｇ加えて溶解し、再度、エタノール（純度９９％）を８０ｇ加えてリン酸化プルランを沈殿させ、得られた沈殿物を６０℃で３時間減圧乾燥し、粉砕して、水分含量が５．２質量％である精製リン酸化プルラン粉末を約２０ｇ得た。
<Preparation of phosphorylated pullulan>
205 g of purified water was added to 45 g of pullulan (trade name “Pullan PI20”, Hayashibara Shoji Co., Ltd., number average molecular weight 110,000 daltons) and dissolved with sufficient stirring. To this pullulan aqueous solution, 1,000 g of polyphosphoric acid aqueous solution prepared in advance so that the concentration of polyphosphoric acid (sold by Wako Pure Chemical Industries, Ltd.) becomes 1 mol (pH 5.5) was added and mixed, and then the whole amount was vacuum dried. Then, it was dried until the water content became 10.2% by mass. The obtained dried product was heated at 170 ° C. for 5 hours to obtain phosphorylated pullulan. The obtained phosphorylated pullulan was pulverized with a mixer to obtain about 130 g of a phosphorylated pullulan pulverized product. After this pulverized product was dissolved in 3,000 g of purified water, 2,500 g of ethanol (purity 99%) was added to precipitate phosphorylated pullulan. The obtained precipitate was collected by centrifugation (5,000 rpm, 15 minutes), further washed twice with 2,000 g of 50% by mass ethanol aqueous solution, and 100 g of purified water was added to the obtained precipitate. About 130 g of a crude phosphorylated pullulan-containing aqueous solution containing about 25 g of solid matter was obtained. 130 g of ethanol (purity 99%) was added to the crude phosphorylated pullulan-containing aqueous solution to precipitate phosphorylated pullulan, the precipitate was recovered, 120 g of purified water was added and dissolved, and again ethanol (purity 99%) Was added to precipitate phosphorylated pullulan, and the resulting precipitate was dried under reduced pressure at 60 ° C. for 3 hours and pulverized to obtain about 20 g of purified phosphorylated pullulan powder having a water content of 5.2% by mass. .
上記の方法で得られた精製リン酸化プルランを精製水１８０ｇ中に溶解し、実験例１に示す方法により、エンドトキシンを除去し、真空乾燥し、粉砕して、水分含量が３．７質量％である精製リン酸化プルラン標品（標品２）を１８ｇ得た。本標品２をエンドトキシン測定試薬（商品名『リムルスＪテストワコー』、和光純薬工業株式会社販売）で測定したところ、本標品２は、エンドトキシンを０．２５ＥＵ／ｍｌ未満含有し、パイロジェンは検出限界以下であった。なお、実験例２で用いた方法により、本標品２におけるリン含量を測定したところ、精製リン酸化プルラン標品（標品２）１ｇ当り、リンを約９．４ｍｇ含んでいた。本標品２の赤外吸収スペクトルを図３に示す。 The purified phosphorylated pullulan obtained by the above method is dissolved in 180 g of purified water, endotoxin is removed by the method shown in Experimental Example 1, vacuum-dried, pulverized, and the water content is 3.7% by mass. 18 g of a purified phosphorylated pullulan sample (standard 2) was obtained. When this sample 2 was measured with an endotoxin measurement reagent (trade name “Limulus J Test Wako”, sold by Wako Pure Chemical Industries, Ltd.), this sample 2 contained less than 0.25 EU / ml of endotoxin. It was below the detection limit. In addition, when the phosphorus content in this sample 2 was measured by the method used in Experimental Example 2, about 9.4 mg of phosphorus was contained per 1 g of the purified phosphorylated pullulan sample (sample 2). The infrared absorption spectrum of this sample 2 is shown in FIG.
＜チタンへの接着性評価試験＞
実験例１で得たリン酸化プルラン標品（標品１）、実験例３で得たリン酸化プルラン標品（標品２）、及び、対照としてのプルラン（商品名『プルラン』、数平均分子量７０、０００ダルトン、株式会社林林原商事販売）を用いて、チタンへの接着性を調べた。上記リン酸化プルラン標品１、２及び対照のプルランを用いて、それぞれ１０質量％の水溶液を調製した。一方、チタン円板（直径１３ｍｍ、厚み２ｍｍ、株式会社ジーシー製）をそのまま、又は、濃度１モル塩酸中に３０分間浸漬（以下、「塩酸処理」と言う。）した後、精製水で洗浄し、次いで、前記標品１、２及び対照のプルラン水溶液に浸漬した後、直ちに取り出し、６０℃で１時間乾燥させ、チタン円板表面に皮膜（膜厚約２００μｍ）を形成させた。次に、各チタン円板に被覆されたリン酸化プルラン皮膜及びプルラン皮膜のチタンへの接着性を評価するため、これらチタン円板表面にセロハンテープ（商品名『セロテープＣ２５２』、工業積水化学工業株式会社製）（７ｃｍ２）を張り付け、１分間経過後、そのテープを剥し、テープ上にリン酸化プルラン皮膜又はプルラン皮膜が付着してチタン円板から剥離するかどうかを調べた。それらの結果を表１にまとめた。
<Evaluation test for adhesion to titanium>
Phosphorylated pullulan sample (sample 1) obtained in Experimental Example 1, phosphorylated pullulan sample (Sample 2) obtained in Experimental Example 3, and pullulan as a control (trade name “Pullan”, number average molecular weight 70,000 Dalton, Hayashibara Shoji Sales Co., Ltd.) was used to examine the adhesion to titanium. Using the
表１の結果から明らかなとおり、リン酸化プルラン標品１及びリン酸化プルラン標品２は、塩酸処理したチタンに対し良好な接着性を示すことが判明した。また、塩酸処理しなかったチタンに対しては、両標品とも低い接着性を示すことが判明した。一方、対照のプルランは、チタンの塩酸処理の有無に関わらず、チタンへの接着性は劣ることも判明した。
As is clear from the results in Table 1, it was found that phosphorylated
以上の結果から、リン酸化プルランは、塩酸処理したチタンに対し良好な接着性を示すことが判明した。 From the above results, it was found that phosphorylated pullulan exhibits good adhesion to hydrochloric acid-treated titanium.
＜細胞接着性＞
マウス骨芽細胞様細胞ＭＣ３Ｔ３−Ｅ１株（ＡＴＣＣ ＣＲＬ−２５９３）を１０％（ｖ／ｖ）牛胎児血清及び７．５質量％ＮａＨＣＯ３を含むα−ＭＥＭ培地（ＩＣＮ Ｂｉｏｍｅｄｉｃａｌ販売）中に１．５×１０５個／ｍｌとなるように懸濁し、３７℃、５％（ｖ／ｖ）ＣＯ２インキュベータ中で７２時間培養した。培養後、細胞を回収し、前記と同様の新鮮な培地中に５×１０５個／ｍｌとなるように懸濁して、細胞懸濁液を調製した。一方、実験例１の方法で調製し、精密濾過して無菌としたリン酸化プルランを、無菌的に塩酸処理したチタン円板（直径１３ｍｍ、厚み２ｍｍ、株式会社ジーシー製）表面に実験例４の方法にしたがって被覆し、無菌精製水５０ｍｌ中に前記チタン円板を浸漬し、直ちに取り出して、上記細胞懸濁液（１ｍｌ）中に浸漬し、３７℃、５％（ｖ／ｖ）ＣＯ２インキュベータ中で１０分間培養した。培養後、チタン円板を培地中から取り出し、リン酸緩衝液（ＰＢＳ）で洗浄してチタン円板に非吸着の細胞を除去し、チタン円板に吸着した細胞をＭＴＳアッセイ試薬（商品名『ＣｅｌｌＴｉｔｅｒ ９６Ｒ』、プロメガ株式会社販売）を用いて測定した。一方、対照として、塩酸処理していないチタン円板（以下、「塩酸無処理チタン」と言う。）を用いた以外は前記同様にしてリン酸化プルランを被覆したチタン円板（対照１）と、塩酸処理のみを施し、リン酸化プルランを被覆していないチタン円板（対照２）を用いて、前記同様にして、細胞を培養し、チタン円板に接着した細胞数を測定したところ、塩酸処理チタン表面にリン酸化プルランを被覆したチタン円板には、２．７×１０５個の細胞が接着していたのに対し、対照１の塩酸無処理チタン表面にリン酸化プルランを被覆したチタン円板には、１．８×１０５個の細胞が接着し、対照２の塩酸処理チタン表面にリン酸化プルランを被覆していないチタン円板には、１．０×１０５個の細胞が接着していた。
<Cell adhesion>
Mouse osteoblast-like cell strain MC3T3-E1 (ATCC CRL-2593) was added to α-MEM medium (sold by ICN Biomedical) containing 10% (v / v) fetal calf serum and 7.5% by mass NaHCO 3 . The suspension was suspended at 5 × 10 5 cells / ml, and cultured in a 5% (v / v) CO 2 incubator at 37 ° C. for 72 hours. After the cultivation, the cells were collected and suspended in a fresh medium similar to the above to a concentration of 5 × 10 5 cells / ml to prepare a cell suspension. On the other hand, the phosphorylated pullulan prepared by the method of Experimental Example 1 and sterilized by microfiltration was applied to the surface of a titanium disc (diameter 13 mm, thickness 2 mm, manufactured by GC Corporation) aseptically treated with hydrochloric acid. Coat according to the method, immerse the titanium disc in 50 ml of sterile purified water, immediately remove it, immerse in the cell suspension (1 ml), 37 ° C., 5% (v / v) CO 2 incubator Incubated for 10 minutes. After incubation, the titanium disk is removed from the medium, washed with phosphate buffer (PBS) to remove non-adsorbed cells on the titanium disk, and the cells adsorbed on the titanium disk are removed from the MTS assay reagent (trade name “ CellTiter 96R ”, sold by Promega Corporation). On the other hand, as a control, a titanium disc (Control 1) coated with phosphorylated pullulan in the same manner as described above except that a titanium disc not treated with hydrochloric acid (hereinafter referred to as “titanium without hydrochloric acid treatment”) was used, When a cell was cultured and the number of cells adhered to the titanium disc was measured in the same manner as described above using a titanium disc (control 2) that was treated only with hydrochloric acid and not coated with phosphorylated pullulan, The titanium disk coated with phosphorylated pullulan on the titanium surface had 2.7 × 10 5 cells adhered to it, whereas the titanium circle coated with phosphorylated pullulan on the surface of hydrochloric acid-untreated titanium of
以上の結果から、塩酸処理チタン表面にリン酸化プルランを被覆したチタンは、対照１、２と比べ、細胞の接着性が有意に高いことが判明した。この事実は、塩酸処理チタン表面にリン酸化プルランを被覆したチタンは、これを口腔インプラント材として用いたとき、適用部位の生体組織との適合性が優れていることを示すものである。
From the above results, it was found that titanium coated with phosphorylated pullulan on the surface of hydrochloric acid-treated titanium had significantly higher cell adhesion than
＜口腔チタンインプラント材＞
実験例１で得たリン酸化プルラン標品（標品１）又は実験例３で得たリン酸化プルラン標品（標品２）を用いて、１５質量％の水溶液を調製した。一方、チタン円柱（直径５ｍｍ、長さ２０ｍｍ）２本を濃度１モル塩酸中に６０分間浸漬した後、精製水で洗浄し、次いで、前記プルラン水溶液のそれぞれに浸漬した後、直ちに取り出し、６０℃で１時間乾燥させ、チタン円柱表面に皮膜（膜厚約２５０μｍ）を形成させ、本発明の口腔チタンインプラント材を二種類得た。本品は、細胞の接着性が高く、口腔インプラント材として用いたとき、適用部位の生体組織との適合性にも優れている。
<Oral titanium implant material>
A 15% by mass aqueous solution was prepared using the phosphorylated pullulan sample (standard 1) obtained in Experimental Example 1 or the phosphorylated pullulan sample (Standard 2) obtained in Experimental Example 3. On the other hand, two titanium cylinders (diameter 5 mm, length 20 mm) were immersed in 1 molar hydrochloric acid for 60 minutes, washed with purified water, then immersed in each of the aqueous pullulan solutions, and then immediately taken out to 60 ° C. And dried for 1 hour to form a film (film thickness of about 250 μm) on the surface of the titanium cylinder, and two types of oral titanium implant materials of the present invention were obtained. This product has high cell adhesion, and when used as an oral implant material, it is also excellent in compatibility with the living tissue of the application site.
＜口腔チタンインプラント材＞
実験例１の方法で得たリン酸化プルラン標品６００ｍｇを精製水３０ｍｌに溶解し、温度５℃に調整した。一方、予め、５ｍｇの塩化シアヌル（和光純薬工業株式会社販売）をアセトン１ｍｌに溶解して得られた塩化シアヌル溶液を、上記リン酸化プルラン含有水溶液に加え、温度５℃に保ちながら攪拌しつつ、濃度３０質量％の炭酸ナトリウム水溶液を用いてｐＨを７．０に調整しながら１時間反応させ、活性化リン酸化プルラン溶液を得、精密濾過して、無菌化した。この活性化リン酸化プルラン溶液２０ｍｌに、予め、塩基性線維芽細胞生長因子（和光純薬工業株式会社販売）５ｍｇをリン酸緩衝液（ｐＨ７．０）１０ｍｌに溶解しておいた塩基性線維芽細胞生長因子溶液を加え、３７℃で５時間攪拌し、結合反応を行い、更に、余分な活性基を保護するためグリシンを６ｇ加え、５℃で８時間攪拌し、線維芽細胞生長因子を結合させたリン酸化プルラン含有液を得た。当該溶液を、ゲル濾過樹脂（商品名『セファデックスＧ１００』、アマシャムバイオサイエンス株式会社販売）６００ｍｌを用いたゲル濾過カラムクロマトグラフィーで分離し、カラムから溶出した溶出液中のリン酸化プルラン量をアンスロン法で、塩基性線維芽細胞生長因子量をイムノアッセイキット（商品名『線維芽細胞成長因子、塩基性、ヒトアナライザ イムノアッセイキット』、和光純薬工業株式会社販売）で測定し、リン酸化プルラン塩基性線維芽細胞成長因子複合体画分を回収した。得られた画分を回収し、これを常法に従って凍結乾燥して、リン酸化プルラン塩基性線維芽細胞成長因子複合体粉末を約３５０ｍｇ得た。本粉末をイムノアッセイに供したところ、本粉末は、粉末１ｇ当り約３．１ｍｇの塩基性線維芽細胞成長因子を含有していることが判明した。本粉末を精製水に溶解して、２０質量％の水溶液を調製した。この水溶液に、チタン円柱（直径７ｍｍ、長さ１２ｍｍ）を濃度２モル塩酸中に２０分間浸漬した後、精製水で洗浄し、次いで、前記プルラン水溶液のそれぞれに浸漬した後、直ちに取り出し、５５℃で１時間乾燥させ、チタン円柱表面に皮膜（膜厚約３００μｍ）を形成させ、本発明の口腔チタンインプラント材を得た。本品は、細胞の接着性が高く、口腔インプラント材として用いたとき、適用部位の生体組織との適合性にも優れている。
<Oral titanium implant material>
600 mg of phosphorylated pullulan sample obtained by the method of Experimental Example 1 was dissolved in 30 ml of purified water and adjusted to a temperature of 5 ° C. On the other hand, a cyanuric chloride solution obtained by dissolving 5 mg of cyanuric chloride (available from Wako Pure Chemical Industries, Ltd.) in 1 ml of acetone in advance is added to the phosphorylated pullulan-containing aqueous solution and stirred while maintaining the temperature at 5 ° C. The mixture was reacted for 1 hour while adjusting the pH to 7.0 using an aqueous sodium carbonate solution having a concentration of 30% by mass to obtain an activated phosphorylated pullulan solution, which was sterilized by microfiltration. Basic fibroblasts in which 5 mg of basic fibroblast growth factor (sales by Wako Pure Chemical Industries, Ltd.) was previously dissolved in 10 ml of phosphate buffer (pH 7.0) in 20 ml of this activated phosphorylated pullulan solution. Add cell growth factor solution, stir at 37 ° C. for 5 hours, perform binding reaction, add 6 g of glycine to protect excess active groups, and stir at 5 ° C. for 8 hours to bind fibroblast growth factor A phosphorylated pullulan-containing liquid was obtained. The solution was separated by gel filtration column chromatography using 600 ml of gel filtration resin (trade name “Sephadex G100”, sold by Amersham Biosciences), and the amount of phosphorylated pullulan in the eluate eluted from the column was determined to be anthrone. The amount of basic fibroblast growth factor was measured with an immunoassay kit (trade name “Fibroblast Growth Factor, Basic, Human Analyzer Immunoassay Kit”, sold by Wako Pure Chemical Industries, Ltd.), and phosphorylated pullulan basic The fibroblast growth factor complex fraction was collected. The obtained fraction was collected and lyophilized according to a conventional method to obtain about 350 mg of phosphorylated pullulan basic fibroblast growth factor complex powder. When this powder was subjected to an immunoassay, the powder was found to contain about 3.1 mg of basic fibroblast growth factor per gram of powder. This powder was dissolved in purified water to prepare a 20% by mass aqueous solution. In this aqueous solution, a titanium cylinder (diameter 7 mm, length 12 mm) was immersed in hydrochloric acid having a concentration of 2 mol for 20 minutes, washed with purified water, then immersed in each of the aqueous pullulan solutions, and then immediately taken out. And dried for 1 hour to form a film (film thickness of about 300 μm) on the surface of the titanium cylinder, to obtain an oral titanium implant material of the present invention. This product has high cell adhesion, and when used as an oral implant material, it is also excellent in compatibility with the living tissue of the application site.
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| BR112019021396A2 (en) | 2017-04-14 | 2020-04-28 | Capsugel Belgium Nv | pullulan manufacturing process |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
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